Biology subjects

Nobrega, M.

Publications and source records attributed to Nobrega, M..

2 recordsLinked to original sources

bioRxiv2018

TCF7L2 Regulation of GATA6-dependent and -Independent Vascular Smooth Muscle Cell Plasticity and Intimal Hyperplasia

Genetic variations in Wnt-coreceptor LRP6 and Wnt-regulated transcription factor TCF7L2 have been among the strongest genetic signals for type2 diabetes (T2DM) and coronary artery disease (CAD). Mice with a CAD-linked LRP6 mutation exhibit obstructive coronary artery disease characterized by reduced TCF7L2 expression and dedifferentiation of vascular smooth muscle cell (VSMC). While TCF7L2 maintains stemness and promotes proliferation in embryonic tissues and adult stem cells, its role and mechanisms of action in VSMC differentiation is not understood. Using multiple mouse models, we demonstrate here that TCF7L2 promotes differentiation and inhibits proliferation of VSMCs. TCF7L2 accomplishes these effects by stabilization of GATA6 and upregulation of SM-MHC and cell cycle inhibitors. Accordingly, TCF7L2 haploinsufficient mice exhibited increased susceptibility to, while mice overexpressing TCF7L2 were protected against injury-induced intimal hyperplasia compared to wildtype littermates. Consequently, the overexpression of TCF7L2 in LRP6 mutant mice rescued the injury induced intimal hyperplasia. These novel findings imply cell type-specific functional role of TCF7L2 and provide critical insight into poorly understood mechanisms underlying pathogenesis of intimal hyperplasia.

molecular biology↗

bioRxiv2016

Reducing mitochondrial reads in ATAC-seq using CRISPR/Cas9

ATAC-seq is a high-throughput sequencing technique that aims at identifying DNA sequences located in open chromatin. Depending on the cell type, ATAC-seq may yield a high number of mitochondrial sequencing reads (~20-80% of the reads). As the regions of open chromatin of interest are usually located in the nuclear genome, mitochondrial reads are typically discarded from the analysis. To decrease wasted sequencing, we performed targeted cleavage of mitochondrial DNA using CRISPR/Cas9 and 100 mtDNA-specific guide RNAs. We also tested a modified ATAC-seq protocol that does not include detergent in the cell lysis buffer. Both treatments resulted in considerable reduction of mitochondrial reads (1.7 and 3-fold, respectively). The removal of detergent, however, resulted in increased background and fewer peaks identified. The highest number of peaks and highest quality data was obtained by preparing samples with the original ATAC-seq protocol (using detergent) and treating them with anti-mitochondrial guide RNAs and Cas9. This strategy could lead to considerable cost reduction and improved peak calling when performing ATAC-seq on a moderate to large number of samples and in cell types that contain a large amount of mitochondria.

genomics↗